A colony of honey bees, Apis mellifera, consists of a single queen, who is usually the mother of all other colony members, 10,000-30,000 semi-sterile female workers, and from zero to a few thousand males (drones), depending on the time of year. Eggs, larvae, and pupae are collectively referred to as brood. Female adult workers perform all of the behavioral tasks associated with colony growth and maintenance. Worker honey bees perform different tasks as they age, a phenomenon referred to as division of labor or temporal polyethism (Robinson 1992; Anderson and Franks 2001). When worker honey bees emerge from their cells as adults their first “job” is normally to clean cells. As the workers age they take on a succession of other jobs: feeding larvae, processing and storing food, secreting wax and constructing comb, and guarding the entrance to the hive. The most obvious change in behavior occurs when worker bees are about three weeks old, and they begin foraging (Lindauer 1952; Seeley and Kolmes 1991). Foraging labor is also divided, whereby some workers return to the colony carrying only nectar, some carry only pollen, and some return carrying both nectar and pollen. Pollen and nectar are sources of protein and carbohydrate, respectively.
Honey bee foragers collect pollen from available plant sources. They then return to the nest and deposit their loads of pollen directly into cells. Stored pollen is consumed by young nurse bees that use the proteins derived from the pollen to produce proteinaceous hypopharyngeal gland secretions that are fed to developing larvae (Crailsheim et al. 1992). By consuming pollen in this manner, nurse bees reduce the quantities of stored pollen. The presence of young larvae affects the proportion of foragers collecting pollen. The more larvae in a colony, the more pollen collected by foraging workers (Al-Tikrity et al. 1972; Barker 1971; Free 1967, 1979; Jaycox 1970; Todd and Reed 1970).
The hypothesis that larval honey bees produce a pheromone that stimulates foraging (Filmer 1932: Free 1967) was tested and verified by Pankiw et al. (1998), who found a 2.5 fold increase in the number of pollen foragers when colonies with 1,000 larvae were provided with hexane extracts of 2,000 honey bee larvae. Pankiw et al. (1998) also found that the same hexane larval extract stimulated pollen foraging in colonies with no larvae, i.e. the extract replaced the larvae as a source of pheromone. Thus, bees apparently determine the number of larvae in a colony by sensing the concentration of chemicals in brood pheromone produced by the larvae.
Pankiw and Page (2001) and Page and Pankiw (2002) tested a synthetic pheromone blend that comprised ten non-volatile esters of fatty acids (LeConte et al. 1990) in the following percentages: ethyl linoleate 1%, ethyl linolenate 13%, ethyl oleate 8%, ethyl palmitate 3%, ethyl stearate 7%, methyl linoleate 2%, methyl linolenate 21%, methyl oleate 25%, methyl palmitate 3% and methyl stearate 17%. This blend was found to lower the neurosensory response threshold to sucrose in worker bees, which is a measure of propensity to forage for pollen. Worker honey bees with low sucrose response thresholds are most likely to forage for pollen, and those with high response thresholds are most likely to forage for nectar (Pankiw and Page 2000; Pankiw 2003; Pankiw et al. 2004a). Within one hour of being placed in the center of a colony, larval extract or the synthetic brood pheromone blend increased the ratio of pollen to non-pollen foragers of colonies in an almond orchard. Based on their results, Pankiw and Page (2001) suggest that “brood pheromone modulates response threshold in pre-foragers, primes them to be pollen foragers, and releases pollen foraging behavior.” It is not known whether different races of honey bees produce pheromone with different ratios of the ten components, or whether worker bees respond preferentially to the blend of pheromone components produced by larvae of their own race.
In a subsequent study, Pankiw et al. (2004b) exposed honey bee colonies to 2,000 larval equivalents (1.12 mg) of synthetic brood pheromone daily for 28 days. As well as increasing the number of pollen foragers, as predicted by earlier studies, brood pheromone had several other important effects. Compared to untreated control colonies, the weights of pollen loads returned to the hive were significantly greater in colonies treated with brood pheromone. Pheromone-treated colonies reared significantly more brood, as measured by the area of comb housing eggs, larvae and pupae. The number of adult honey bees that emerged from this brood was significantly greater in colonies treated with brood pheromone than in untreated control colonies. Thus the overall size of colonies was increased.
Brood pheromone can have dose-dependent effects (Pankiw 2004a; LeConte et al. 2000). At a low, biologically realistic dose of 2,000 larval equivalents, brood pheromone recruited honey bee workers to become foragers at an earlier age than in control colonies (Pankiw et al. 2004), whereas at a much higher dose of 6,200 larval equivalents, brood pheromone delayed the recruitment of workers to become foragers (LeConte et al. 2000).
Pankiw et al. (2004) found that although exposure to brood pheromone generally lowered the age of first foraging, the onset of foraging was delayed in a specialized cohort of worker bees that had a very high content of protein in their hypopharyngeal glands. This high protein level is an indication of increased probability of brood rearing behavior and a correspondingly higher quality of protein fed to larvae compared to that in control colonies.
Pankiw (2004b) found that the ratio of pollen foragers to non-pollen foragers increased within one hour of placement of 2,000 larval equivalents of synthetic brood pheromone into a honey bee colony. Foragers from pheromone-treated colonies returned with heavier pollen loads than foragers from control colonies, and this pollen was 43% more likely to originate from the target crop within which colonies were placed to ensure pollination. Non-pollen foragers, that may visit and pollinate more flowers than pollen foragers while searching for the best nectar sources, had more pollen grains on their bodies than non-pollen foragers from untreated control colonies. Similar to pollen foragers, these non-pollen foragers bore pollen that was 54% more likely to originate from the target crop than pollen borne by non-pollen foragers from control colonies. By increasing the activity of both pollen foragers and non-pollen foragers, brood pheromone has the potential to greatly increase the effectiveness of colonies used in custom pollination of target crops.
Another potential use for brood pheromone is stimulating the consumption of dietary protein supplements. Beekeepers commonly provide a protein supplement in winter or early spring to promote colony growth (Herbert 1992). Often an antibiotic is blended with the protein supplement as a prophylactic against larval bacterial diseases. However, because larvae are absent or at their lowest levels in late winter and early spring, there is very little, if any, brood pheromone being produced within the colony that might stimulate the consumption of protein and antibiotic dietary supplements, as well as hypopharyngeal gland development and protein biosynthesis. Administration of synthetic brood pheromone within a colony could provide the necessary stimulus.
Despite the practical promise of brood pheromone (Pankiw 2004b), no commercial products have been developed that incorporate its ten components, apparently because of their instability at room temperature. In fact, Page and Pankiw (2002) teach that the “synthetic brood pheromone is easily oxidizable, and must be stored in low-oxygen conditions, preferably at −20° C., and most preferably at −70° C., if it will be stored for any long period of time.” Even freezing at −70° C. may not stabilize the pheromone. For example, crude honey bee larval extract lost all pheromonal activity after only three months in a laboratory freezer (T. Pankiw, Texas A&M University, unpublished observation). Thus any commercial product that incorporated either the natural or synthetic pheromone would have almost no shelf life, and would potentially lose all potency during storage and shipping. Freezing at would be prohibitively expensive, and would probably not prolong the shelf life for an acceptable duration. Recognizing the instability of the synthetic pheromone, researchers have invariably provided their experimental bees with fresh pheromone each day, usually on glass plates (Pankiw et al. 1998; Pankiw and Page 2001; Page and Pankiw 2002; Pankiw and Rubink 2002; Pankiw et al. 2004b). In commercial operational use, daily application of pheromone would be impractical and expensive, and would not eliminate the need to freeze the pheromone during storage.
Two main hypotheses could potentially explain the short duration of bioactivity of the synthetic and natural pheromone: 1) minute amounts of breakdown products have a positive effect at first, but over time rise to inhibitory levels, or 2) the fresh pheromone is biologically active, but some or all of its constituents break down rapidly to sub-threshold levels. If the synthetic pheromone composition could be stabilized, and if it were then inactive when experimentally tested, the results would support the first hypothesis. If the synthetic pheromone composition were stabilized and remained active in an experimental test, the results would uphold the second hypothesis. The first hypothesis appears most likely, as it is supported by the fact that freshly prepared synthetic brood pheromone can have dose-dependent effects, with high doses negating the positive effect seen with low doses (Pankiw 2004a; LeConte et al. 2000). In this case, there would be no practical purpose in stabilizing the pheromone. On the other hand, if the second hypothesis were upheld, the stabilized synthetic pheromone would have practical utility.
However, if the stabilization process involved adding a chemical stabilizer, such as an antioxidant, the stabilizer could potentially cause the synthetic pheromone composition to become inactive. This could occur through denaturing one or more of the essential components in a complex pheromone blend. It could also inhibit the pheromone receptor membrane on the antennal or palpal sensilla, causing the insect not to perceive the pheromonal message. Finally, employing an altered composition could cause an insect to perceive the “wrong” pheromone blend and thus not to respond.
Antioxidants are not naturally occurring in honey bee colonies. The long-term storage of honey bee food does not involve antioxidants, but relies instead on other methods of preservation. For example, the enzymes diastase, invertase, and glucose oxidase are added by the bees to nectar soon after it is deposited into cells. Glucose oxidase breaks down glucose and releases hydrogen peroxide, an antibacterial agent of anaerobic bacteria. The water content of honey is reduced to less than 18%. This supersaturated solution of sucrose inhibits fungal growth. Fermentation is negated by preventing re-hydration by sealing honey with a thin sheet of wax. Stored pollen is preserved using honey. The enzymes in the honey prevent pollen germination and microbial growth.
Even if it were conceivable to add an antioxidant to a fatty acid pheromone, it is not evident to a person skilled in the pheromone art that the bioactivity of the pheromones would be retained with the addition of an antioxidant. The bioactivity of pheromones is very sensitive and unpredictable. The addition of a foreign substance to a complex pheromone blend is counterintuitive to a person skilled in the art of pheromone biology. To such a person, the addition of an antioxidant might just as easily have negative rather than positive consequences on honey bee foraging behaviors and physiological effects. For example, an antioxidant could prove to be repellent or toxic to honey bees, negating any positive effect of the formulated pheromone. In addition, bioactivity could reside in part in minute amounts of breakdown products, that later accumulate to inhibitory levels, and that would not be produced in the presence of an antioxidant. The relevance of the antioxidant is not centered on chemical stability alone, but rests also on maintaining the behavioral and physiological responses to the pheromone in the presence of an antioxidant that is not naturally occurring with the pheromone or a honey bee colony.
Doisaki et al. (2006), Quimica Nova (2006), and Critical Reviews in Food Science and Nutrition (1995) disclose antioxidants but do not deal with pheromones. The disclosed antioxidants can be used as additives for fats and oils to enhance their oxidative stability. There is no teaching in any of these references that the disclosed antioxidants can be used with pheromones. The respective arts of pheromones and stabilizing fats and oils with antioxidants are distinct and not related. A person skilled in the art of pheromones is not likely to look to the other art for inspiration.